Trace and Ultra-Trace Analysis
Environmental protection agency defines trace analysis as analysis at part-per-thousand level (http://www.epagov/esd/chemistry/org-anal/faq.htm).
According to IUPAC definition, trace analysis corresponds to analysis of compounds below 100 ppm (<0.01%). Microtrace analysis corresponds to analysis below 1 ppm (<10−4%), ultra-microtrace analysis to analysis below 1 ppb (<10−7%), and sub-microtrace analysis to analysis below 1 ppt (<10−10%) (J. Namiesnik Trace Analysis—Challenges and Problems. Crit. Rev. Anal. Chem. 32, 271-300, 2002).
Separation in Isotachophoresis
In isotachophoresis (ITP), the concentration of ions in their pure isotachophoretic zones, is set up by their mobilities, the mobilities of the used leading ion and counter ion and the concentration of the leading ion and is independent of the original concentration of analytes in the sample. Thus, low-concentration analytes are concentrated between leading ion and terminating ion, which, in combination with a suitable detection method, makes it a useful tool for a rapid ion analysis. To achieve a full isotachophoretic separation of analytes, a certain amount of electric charge has to pass through the separation column (Boc̆ek, P., et al., Analytical isotachophoresis: The concept of the separation capacity. J. Chromatogr., 1979, 160, 1-9.). When so-called effective mobilities of analytes are known, the separation charge and column charge/hold-up can be calculated (Dolník, V., et al., Optimization of isotachophoretic analysis: use of the charge-based transient state model. J. Chromatogr., 1991, 55, 249-266.)
Increasing Separation Performance in Isotachophoresis
Complex mixtures require larger separation charge to achieve full separation of all analytes. Isotachophoretic separation requiring a large separation charge in a narrow capillary would need rather impractically long separation times. Various techniques were introduced to provide high separation charge in short separation time, including hydrodynamic counter flow, volume coupling, column coupling, concentration cascade/step, and multicolumn ITP.
Hydrodynamic counter flow allows performing isotachophoretic separation in relatively short separation column (F. M. Everaerts, Th. P. E .M. Verheggen, J. L. M. Van De Venne: Isotachophoretic experiments with a counter flow of electrolyte. J. Chromatogr. A, 1976, 123, 1139-148).
The volume coupling is a method, where the isotachophoretic separation is performed in a wide separation channel and detection in a narrow detection channel. (Th. P. E. M. Verheggen, F. M. Everaerts, Volume-coupling in isotachophoresis. J. Chromatogr. A, 1982, 249, 221-230).
The column coupling is a method similar to volume coupling. At the interface between two columns of different width, an auxiliary electrode is connected allowing application of high electric current in the separation step without overheating (F. M. Everaerts, Th. P. E. M. Verheggen, F. E. P. Mikkers, Determination of substances at low concentrations in complex mixtures by isotachophoresis with column coupling. J. Chromatogr. A, 1979, 169, 21-38).
The column-coupling method was also applied to separation on chip (R. Bodor, et al., Isotahophoresis and isotachophoresis-zone electrophoresis of food additives on as chip with column-coupling separation channels. J. Sep. Sci, 2001, 24, 802-809.)
The concentration cascade/step is a method where separation is performed in a channel with a high-concentration leading electrolyte and detection in a channel with a low-concentration leading electrolyte (Boc̆ek, P., et al., Effect of a concentration cascade of the leading electrolyte on the seperation capacity in isotachophoresis. J. Chromatogr., 1978, 156, 323-326.).
Concentration step and column coupling can also be combined. This approach brings possible additional advantages such as changing migration order and eliminating major interfering compounds (V. Dolník, M. Deml and P. Boc̆ek: Multivalent ion interactions in isotachophoresis and their utilization in a cascade system. In: C. J. Holloway, (Ed.), Analytical and Preparative Isotachophoresis, Walter de Gruyter, Berlin-N.Y., 1984, pp. 55-62.).
The multicolumn isotachophoresis is a method similar to the column coupling. The separation is performed in a channel with a cross section substantially exceeding the cross-section of the analytical capillary (Dolnik, V., et al., Large sample volume preseparation for trace analysis in isotachophoresis. J. Chromatogr, 1985, 320, 89-97, Dolnik, V., et al., Determination of oxalate in human serum by multicolumn isotachophoresis. Electrophoresis, 1988, 9, 839-841.). To improve dissipation of generated Joule heat, the separation channel has rectangular cross-section as well as the following tapered channel, in which the profile of isotachophoretic zones is reduced. Only a single auxiliary electrode is connected to the tapered channel. During migration through the tapered channel, the fully separated isotachophoretic zones can be partly mixed and have to be restored by migration through a narrow restoring channel. The system may contain a series of tapered and corresponding restoring channels. The final restoring channel serves also as an analytical channel.
Detection in Capillary Electrophoresis
To detect UV absorbing analytes, UV absorption detection can be applied (J. W. Jorgenson, K. DeArman Lukacs: Zone electrophoresis in open-tubular glass capillaries. Anal. Chem., 1981, 53 ,1298-1302, M. T. Ackermans, F. M. Everaerts, J. L. Beckers: Determination of aminoglycoside antibiotics in pharmaceuticals by capillary zone electrophoresis with indirect UV detection coupled with micellar electrokinetic capillary chromatography. J. Chromatogr. A, 1992, 606, 228-235.
For a sensitive detection of fluorescent compounds, fluorescence detection has been applied (J. C. Reijenga, Th. P. E. M. Verheggen, F. M. Everaerts: Fluorescence emission and fluorescence quenching as detection methods in isotachophoresis. J. Chromatogr. A, 1984, 203, 99-111). Laser induced fluorescence detection further improves the detection sensitivity (J. V. Sweedler, J. B. Shear, H. A. Fishman, Richard N. Zare, R. H. Scheller: Fluorescence detection in capillary zone electrophoresis using a charge-coupled device with time-delayed integration. Anal. Chem., 1991, 63, 496-502, X. C. Huang, M. A. Quesada, R. A. Mathies: Capillary array electrophoresis using laser-excited confocal fluorescence detection. Anal. Chem., 1992, 64, 967-972).
Mass spectrometry has been used to detect zones in CZE (R. D. Smith, C. J. Barinaga H. R. Udseth: Improved electrospray ionization interface for capillary zone electrophoresis-mass spectrometry. Anal. Chem., 1988, 60, 1948-1952.)
Nuclear magnetic resonance has been also use for detection of analytes in CZE. (D. L. Olson, M. E. Lacey, A. G. Webb, and J. V. Sweedler: Nanoliter-Volume 1H NMR Detection Using Periodic Stopped-Flow Capillary Electrophoresis. Anal. Chem., 1999, 71, 3070-3076.)
Analytes that do not provide a specific signal can be detected by potential gradient detection (M. Deml, P. Boc̆ek, J. Janak: High-speed isotachophoresis: current supply and detection system. J. Chromatogr. A, 1975, 109, 49-55) and conductivity detection (F. M. Everaerts, Th. P. E. M. Verheggen: Isotachophoresis: Applications in the biochemical field. J. Chromatogr. A, 1974, 91, 837-851). Recently capacitively coupled contactless conductivity detection (C4D) has been applied particularly to CZE (A. Zemann, E. Schnell, D. Volgger, and G. K. Bonn: Contactless Conductivity Detection for Capillary Electrophoresis. Anal. Chem., 1998, 70, 563-567, J. A. Fracassi da Silva and C. L. do Lago: An Oscillometric Detector for Capillary Electrophoresis. Anal. Chem., 1998, 70, 4339-4343, J. Tanyanyiwa and P. C. Hauser: High-voltage contactless conductivity detection of metal ions in capillary electrophoresis. Electrophoresis, 2002, 23, 3781-3786.)
Combining Isotachophoresis and Zone Electrophoresis
It may be advantageous to combine isotachophoresis concentrating analytes with zone electrophoresis and more sensitive detection and quantitation. Disc electrophoresis was, in principle, combination of ITP and zone electrophoresis without online detection (L. Ornstein. Disc electrophoresis: Background and theory. Ann. N.Y. Acad. Sci., 1964, 121, 321-349, B. J. Davis. Disc electrophoresis: II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 1964, 121, 404-427).
In isotachophoresis, quantitative analysis is typically performed by measuring the length of isotachophoretic zones (Boc̆ek, P., et al., Analytical Isotachophoresis. 1988, Weinheim: VCH Publisher.). This translates into limited detection sensitivity. Thus, a combination of ITP and capillary zone electrophoresis (CZE) has been introduced, where analytes are concentrated and separated by ITP and detected by CZE (Pantůc̆kováP., et al., Determination of iodide on samples with complex matrices by hyphenation of capillary isotahcophoresis and zone electrophoresis. Electrophoresis, 2007, 28, 3777-3785.). The ITP-CZE combined analysis was also performed on microchip (Kaniansky D, Masar M, Bielcikova J, Ivanyi F, Eisenbeiss F, Stanislawski B, Grass B, Neyer A, Johnck M. Capillary electrophoresis separations on a planar chip with the column-coupling configuration of the separation channels. Anal Chem. 2000 72, 3596-604, 2000).
Coatings to Suppress Electroosmotic Flow
Electroosmotic flow (EOF) is generated when an electric field is applied to electrolyte in a column that exhibits some electric charged on its inner surface. EOF is detrimental to high-resolutions separations and has to be eliminated.
Chiari (U.S. Pat. No. 6,410,668) disclosed copolymers of various derivatives of acrylamide and methacrylamide with glycidyl group containing monomers to form a highly hydrophilic, dynamic coating that suppresses electroosmotic flow.
Madabhushi et al. (U.S. Pat. No. 5,567,292) disclosed copolymersfor coating suppressing EOF, selected from the group consisting of polylactams, such as poly(vinyl pyrrolidone); N,N-disubstituted polyacrylamides; and N-substituted polyacrylamides.
Dolnik (U.S. Pat. No. 7,799,195) disclosed a permanent wall coating made of thermally immobilized galactomannans to eliminate electroosmotic flow.